Project Summary ? Abstract Multifunctional, nanostructural-based, tissue-engineering technologies have the potential to provide a more effective approach to bone regeneration that will speed healing, improve patients' chances of recovery from debilitating injuries and diseases, and return normal form and function. We hypothesize that in order to improve clinical outcomes in the use of nanomaterials and mesenchymal stem cells (MSCs), it is critical to evaluate the signaling pathways that are triggered when cells adhere to nanomaterials and also to monitor the temporal and spatial changes associated with these processes. Our team of UTK and UALR investigators have identified a system in which a self-supporting, nanocomposite containing low oxygen-functionalized form of graphene (LOGNC) exerts spontaneous osteoinductive and osteoconductive effects on MSCs in vitro and in vivo. Our data supports the notion that human mesenchymal stem cells adhere to the nanocomposites via fibronectin/integrin binding which then influences the subsequent osteogenic response. This system provides us with an opportunity to evaluate the effect of graphene nanocomposites on this process without the involvement of any other exogenous bioactive factor. We therefore propose two Specific Aims: 1) To assess cell adhesion, proliferation, and osteogenic differentiation of human MSCs on 2D and 3D LOGNCs in vitro; 2) To evaluate the biocompatibility and therapeutic potential of human MSCs and 3D LOGNC scaffolds in a bone defect model in vivo. In both aims, we will evaluate the molecular events underlying osteogenesis by using a fluorescent reporter construct under the control of the osteocalcin promoter. We will identify the signaling pathways that are triggered when MSCs adhere and differentiate on LOGNC surfaces. We expect, to understand the interacting mechanisms between MSCs and LOGNCs, to identify at least one combination of MSCs and LOGNC capable of inducing robust osteogenesis, and thus achieve the long-term goal of developing graphene-based nanomaterials that may be implemented in human medicine with the ultimate goal to advance bone regeneration with return to normal form and function for the patient. Moreover, we will gain insight into in vivo physiological processes regarding MSC/scaffold retention, host cell/scaffold infiltration and scaffold degradation. A multicomponent nanoscale graphene-based material could form the foundation for novel scaffold technology to promote rapid bone regeneration to advance bioengineering and promote human health. The proposed project contains basic and clinical components, and, if funded, will create research opportunities for undergraduate and graduate students from biochemistry, cell and molecular biology, materials chemistry, veterinary and human medicine.